Aging-induced increases in pro-inflammatory cytokines IL-1 and IL-18 are associated with the development of several chronic diseases. Notably, recent studies from our research team and other independent laboratories have shown that caloric excess-induced increases in IL-1 and IL-18 are dependent on assembly of a multiprotein signaling platform called the Nlrp3 inflammasome. A novel immune sensor called the Nlrp3 inflammasome controls several aspects of myeloid cell activation and inflammation by controlling the production of IL-1 and IL-18. The protein-protein interactions between Nlrp3 (for nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing-3), the adaptor protein Asc, and procaspase1 lead to the assembly of the Nlrp3 inflammasome and caspase-1 activation. The cysteine protease, caspase-1, is required for the secretion of IL-1 and IL-18 from myeloid-lineage cells. The dietary interventions that dampen excessive inflammasome activation in humans are therefore highly relevant for delaying age-related chronic diseases that stem from inflammation. Caloric restriction remains one of the most robust strategies to extend healthspan and reduce inflammation in animal models. However, bench-to-bedside translation efforts of findings generated from animal studies to humans have been especially challenging in the context of immune cell function and inflammation because the majority of CR studies in inbred animal strains reared in specific pathogen-free facilities do not mimic normal human immune cell activation and deactivation in response to pathogen exposure throughout the lifespan. In addition, key proteins like CARDINAL/CARD8, which are required for the Nlrp3 inflammasome assembly in humans, are absent in mice. Furthermore, inhibitory regulators of inflammasomes and caspase-1, such as ICEBERG and INCA, are present in humans but are absent in worm, fly, or mouse models. Therefore, studies in animal models that evaluate the impact of CR on inflammation are incomplete unless specific inflammasome regulatory mechanisms are studied in humans. The CALERIE-II study is currently evaluating the impact of 2 year CR on human physiology. Using archived immune cell samples from control and CR subjects collected in our lab and adipose tissue biopsies from CALERIE repository, we propose to test the hypothesis that CR-induced deactivation of Nlrp3 inflammasome activation breaks the feed-forward loop of inflammation and prevents metabolic dysfunction in non-obese adults. This project will allow us to assess specific hypotheses and predictions about the relationships between CR and inflammation and role of myeloid cells in adipose-immune crosstalk - in particular the mechanism of Nlrp3 Inflammasome activation - with implications for developing future dietary interventions or pharmacological therapeutic strategies that promote human healthspan.